Name: methods for the modulation and analysis of nf-&#954;b-dependent adult neurogenesis
Uploaded: 2014-02-13T23:00:00
Description: Watch this Scientific Journal Video about Methods for the Modulation and Analysis of NF-ÎºB-dependent Adult Neurogenesis at JoVE.com

We thank Angela Kralemann-K&#246;hler for excellent technical support. Experimental work described herein was performed in our laboratory and was supported by grants of the German Research Council (DFG) to CK and BK&#160;and a grant of the German Ministry of Research and Education (BMBF) to BK.

Ethics statement
This study was carried out in strict accordance with the regulations of the governmental animal and care use committee, LANUV of the state North Rhine-Westphalia, (D&#252;sseldorf, Germany). All animal experiments were approved by LANUV, D&#252;sseldorf under the license number 8.87&#8211;51.04.20.09.317 (LANUV, NRW). All efforts were made to minimize distress and the number of animals required for the study.
1. Animal Care and Housing
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Adult neurogenesis, and the possibility of its manipulation via inhibition of NF-&#954;B in neurons, and its later reactivation via doxycycline,&#160;offers a fascinating system for investigations into newborn neurons in the adult brain, as well as into neuronal de- and re-generation. The beauty of this system is that NF-&#954;B signaling pathway inhibition in neurons not only results in changes in neuronal cell death, progenitor proliferation and migration, and severe structural and anatomical changes, but also in obvio...

Ontologically, the hippocampus is one of the oldest anatomical brain structures known. It is responsible for diverse complex tasks, such as pivotal functions in the regulation of long-term memory, spatial orientation, and formation and consolidation of the respective memory. Anatomically, the hippocampus consists of pyramidal cell layers (stratum pyramidale) including the cornu Ammonis (CA1, CA2, CA3, and CA4) regions and the dentate gyrus (gyrus dentatus), which contains granule cells and a few neuronal progenitors within its subgranular zone. The granule cells project towards the CA3 region via the so-called mossy fibers (axons of granule cells).
Until the end of the last century, the adult mammalian brain was believed to be a static organ lacking cellular plasticity and neurogenesis. However, during the last two decades, a growing amount of evidence clearly demonstrates adult neurogenesis taking place in at least two brain regions, the subventricular zone (SVZ) and the subgranular zone of the hippocampus.
Our previous studies, and those of other groups, have shown that the transcription factor NF-&#954;B is one of the crucial molecular regulators of adult neurogenesis, and that its de-regulation results in severe structural hippocampal defects and cognitive impairments1-6. NF-&#954;B is the generic name of an inducible transcription factor composed of different dimeric combinations of five DNA-binding subunits: p50, p52, c-Rel, RelB, and p65 (RelA), the latter three of which have transactivation domains. Within the brain, the most abundant form found in the cytoplasm is a heterodimer of p50 and p65, which is kept in an inactive form by inhibitor of kappa B (I&#954;B)-proteins.
To study and directly manipulate NF-&#954;B-driven neurogenesis, we use transgenic mouse models to enable simple inhibition of all of the NF-&#954;B subunits, specifically in the forebrain7 (see Figure&#160;1). For this purpose, we cross-bred the following transgenic mouse lines, I&#954;B/- and -/tTA. The transgenic I&#954;B/- line was generated using a trans-dominant negative mutant of NF-&#954;B-inhibitor I&#954;Ba (super-repressor I&#954;Ba-AA1)8. In contrast to the wild-type I&#954;B&#945;, I&#954;B&#945;-AA1 has two serine residues mutated to alanines (V32 and V36), which hinder the phosphorylation and subsequent proteasomal degradation of the inhibitor. For forebrain neuron-specific expression of the I&#954;Ba-AA1-transgene, I&#954;B/- mice were cross-bred with mice harboring a calcium-calmodulin-dependent kinase II&#945; (CAMKII&#945;)-promoter that can be driven by tetracycline trans-activator (tTA)9.
p65 knock-out mice have an embryonic lethal phenotype, due to massive liver apoptosis10, so the approach shown here provides an elegant method for investigating the role of NF-&#954;B in postnatal and adult neurogenesis.
The classic behavioral test to study spatial learning and memory was described in the 1980s by Richard Morris, a test known as the Morris water-maze (MWM)11. In this open-field water-maze, animals learn to escape from opaque water onto a hidden platform based on orientation and extra-maze cues. A dry variant of MWM is the so-called Barnes maze (BM)12. This test utilizes a circular plate with 20 circular holes arranged at the border of a plate, with one defined hole as an escape box, and visual extra-maze cues for orientation. Both experimental paradigms rely on the flight behavior induced by a rodent`s aversion to water, or open, brightly illuminated spaces. Both tests allow an investigation of spatial orientation, and the related memory performance. Although the hippocampus plays a general and essential role in the spatial memory formation, the hippocampal regions involved differ depending on the test applied. The memory tested in BM arises from neuronal activity between the enthorinal cortex (EC) and the pyramidal neurons located in the CA1-region of the hippocampus without a contribution of the DG13-16. In particular, the classic BM mainly relies on navigation via the monosynaptic temporo-ammonic pathway from EC III to CA1 to EC V. Importantly, the DG is crucially involved in the so-called spatial pattern recognition17, which implies not only the processing of visual and spatial information, but also the transformation of similar representations or memories into dissimilar, nonoverlapping representations. This task requires a functional tri-synaptic circuit from EC II to DG to CA3 to CA1 and EC VI, which cannot be tested in the BM15.
To address these challenges, we have devised SPS-BM as a behavioral test to specifically test dentate gyrus-dependent cognitive performance in control animals, and in the I&#954;B/tTA super-repressor model following NF-&#954;B inhibition. Importantly, in contrast to the MWM or the BM, the SPS-BM can reveal subtle behavioral deficits resulting from impairment of neurogenesis. Since spatial-pattern-separation is strictly dependent on a functional circuit between EC II and DG and CA3 and CA1 and EC VI, this test is highly sensitive to potential changes in neurogenesis, modifications of the mossy fiber pathway or alterations of tissue homeostasis within the DG.
Technically, the set-up of our test is based on the study by Clelland et al., in which the spatial separation pattern was tested using a wooden 8-arm radial arm maze (RAM)19. In our modified set-up, the eight arms were replaced by seven identical yellow food houses. In summary, the methods shown here, including analysis of doublecortin-expressing (DCX+) cells within the hippocampus, the mossy fiber projections, neuronal cell death and particularly the SPS-BM presented here, can be applied to investigations of other mouse models incorporating transgenes that have an impact on adult neurogenesis. Further applications may include the study of pharmacological agents and measuring their impact on DG and spatial pattern separation.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Moria MC17 Perforated Spoon&#160;</td>
			<td style="width:127px;">FST</td>
			<td style="width:132px;">10370-18</td>
			<td style="width:167px;">removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting microscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi SV8</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical scissors&#160;</td>
			<td style="width:127px;">FST</td>
			<td>14084-08</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical scissors&#160;</td>
			<td style="width:127px;">FST</td>
			<td>14381-43</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5 forceps</td>
			<td style="width:127px;">FST</td>
			<td>11254-20</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">SuperFrost Slides</td>
			<td>Carl Roth&#160;</td>
			<td align="right">1879</td>
			<td>slides for immunohistochemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Paraformaldehyde</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>P6148</td>
			<td>fixative</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">TissueTek OCT compound</td>
			<td>Sakura Finetek</td>
			<td style="width:132px;">1004200018</td>
			<td>embedding of the brains</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:447px;">Normal Goat Serum</td>
			<td style="width:127px;">Jackson Immunolabs</td>
			<td style="width:132px;">005-000-001</td>
			<td>blocking in IHC</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:447px;">Normal Rabbit Serum</td>
			<td style="width:127px;">Jackson Immunolabs</td>
			<td style="width:132px;">011-000-001</td>
			<td>blocking in IHC</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Normal Donkey Serum</td>
			<td style="width:127px;">Jackson Immunolabs</td>
			<td style="width:132px;">017-000-001</td>
			<td>blocking in IHC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Neurofilament-M antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>2H3</td>
			<td>IHC, Dilution 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-doublecortin&#160;antibody</td>
			<td>sc-8066</td>
			<td>Santa Cruz</td>
			<td>IHC, Dilution 1:800</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">anti-GFP antibody</td>
			<td>ab290</td>
			<td>abcam</td>
			<td>IHC, Dilution 1:2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">anti-BrdU antibody</td>
			<td>OBT0030G</td>
			<td>Accurate Chemicals</td>
			<td>IHC, Dilution 1:2000&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Fluoro Jade-C</td>
			<td>FJ-C</td>
			<td>HistoChem</td>
			<td>Determination of neuronal cell death</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Betadine</td>
			<td style="width:127px;">&#160;MUNDIPHARMA</td>
			<td>D08AG02&#160;</td>
			<td>disinfectant</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cryomicrotome</td>
			<td>Leica</td>
			<td>CM1900</td>
			<td>preparation of brain slices</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Heparin sodium salt</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:132px;">H3393</td>
			<td>perfusion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">circular plate made from hard-plastic (diameter 120 cm)</td>
			<td style="width:127px;">lab made</td>
			<td style="width:132px;">none</td>
			<td>plate for SPS-BM, diameter 120cm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buraton rapid disinfectant&#160;</td>
			<td style="width:127px;">Sch&#252;lke &#38; Mayr</td>
			<td style="width:132px;">113 911</td>
			<td>disinfectant</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">video-tracking system TSE VideoMot 2 with Software Package VideoMot2</td>
			<td>tse systems</td>
			<td style="width:132px;">302050-SW-KIT</td>
			<td>tracking and analysis of SPS-BM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td>permeabilisation/IHC</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:447px;">cryotome</td>
			<td style="width:127px;">Reichert&#160; Jung/Leica</td>
			<td style="width:132px;">Frigomobil 1206</td>
			<td>preparation of 40&#181;m brain slices</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Mowiol 4-88</td>
			<td style="width:127px;">Carl Roth&#160;</td>
			<td>Art.-Nr. 0713</td>
			<td>embedding of the slides</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">SYTOX green</td>
			<td style="width:127px;">Invitrogen</td>
			<td>S7020</td>
			<td>Nuclear staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Food pellets (Kellog`s Froot Loops)</td>
			<td>Kellog`s</td>
			<td style="width:132px;"></td>
			<td>SPS-BM</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:447px;">Prism, Version 3.0</td>
			<td style="width:127px;">Graph Pad Software, San Diego, USA&#160;</td>
			<td style="width:132px;"></td>
			<td>Statistical evaluation of SPS-BM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Zen 2008 or Zen 2011 Software</td>
			<td style="width:127px;">Carl Zeiss</td>
			<td style="width:132px;"></td>
			<td>Software (Confocal microscope)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">D.P.X</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:132px;">317616</td>
			<td>mounting medium for Fluoro Jade C staining</td>
		</tr>
	</tbody>
</table>

methods for the modulation and analysis of nf-&#954;b-dependent adult neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Cross-breeding of the I&#954;B/- and tTA transgenic mouse lines leads to conditional inhibition of NF-&#954;B activity in the hippocampus. 
To investigate the expression of the I&#954;B&#945;-AA1-transgene in the double transgenic mouse (Figure 1A), brains were isolated, cryosectioned and stained using an antibody against GFP (green fluorescent protein). Confocal laser scanning microscopy revealed high expression of the transgene in the CA1 and CA3 regions, an...

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Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

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Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...